Methods for treating cancer in patients with elevated levels of Bim

ABSTRACT

Materials and methods for treating cancer, including materials and methods for treating cancer in patients identified as having elevated levels of Bim.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 15/026,461, filed onMar. 31, 2016 (now U.S. Pat. No. 10,259,875), which is a National Stageapplication under 35 U.S.C. § 371 of International Application No.PCT/US2014/053870, having an International Filing Date of Sep. 3, 2014,which claims benefit of priority from U.S. Provisional Application Ser.No. 61/885,218, filed on Oct. 1, 2013. The disclosures of the priorapplications are considered part of the disclosure of this application,and are each incorporated in their entirety into this application.

TECHNICAL FIELD

This document relates to materials and methods for treating cancer, andmore particularly to materials and methods for treating cancer inpatients identified as having elevated levels of Bim.

BACKGROUND

The metastatic spread of tumor cells is the primary cause of cancerrelated mortality, indicating a need for therapeutic approaches capableof controlling or preventing metastasis (Gibbons et al. (2012)OncoImmunology 1(7):1061-1073; and Grivennikov et al. (2010) Cell140:883-899). The presence of tumor-infiltrating effector and memory Tcells is correlated with decreased metastatic spread, consistent with arole for T cells in preventing metastasis of primary tumors.

B7-H1 (also referred to as PD-L1) is a polypeptide expressed by avariety of tumor cells. It also is constitutively expressed bymacrophages and dendritic cells, and its expression is up-regulated uponcell activation. PD-1 is expressed on the surface of activated T cells,B cells, and macrophages, and is a receptor for B7-H1. CD80 is found onactivated B cells and monocytes, and provides a costimulatory signalnecessary for T cell activation and survival; CD80 also binds B7-H1.

SUMMARY

This document provides, inter alia, a method for determining whetherPD-1 on T cells has engaged its ligand, B7-H1. The method is based inpart on the discovery that engagement of PD-1 by B7-H1 results inup-regulation of Bim, a pro-apoptotic molecule, and is correlated withB7-H1-mediated T cell death. This discovery suggests that intracellularlevels of Bim among PD-1 positive cells is a barometer of the extent towhich PD-1 has been triggered by B7-H1, with lower levels of Bimidentifying activated PD-1 positive T cells whose PD-1 molecules havenot yet been extensively engaged, and higher levels of Bim reflectingchronic engagement of PD-1 with B7-H1. Stratifying Bim levels among PD-1positive CD8 T cells may be a biomarker for gauging (1) whether PD-1molecules on CD8 T cells have been engaged by B7-H1 tumor associatedligands; and (2) the efficacy of an anti-PD-1 or anti-B7-H1 blockaderegimen in reducing PD-1 engagement. Thus, using Bim as a signalingbiomarker for PD-1 function, it may be possible to select patients morelikely to benefit from checkpoint blockade therapy and to identifyoptimal therapeutic timing and dosing schedules.

In one aspect, this document features a method for treating a mammalhaving cancer, wherein said method comprises: (a) identifying saidmammal as containing an elevated level of Bim, and (b) administering tosaid mammal an anti-B7-H1 antibody, an anti-PD-1 antibody, an anti-CD80antibody, a fusion protein comprising a portion of PD-1 linked to animmunoglobulin Fc sequence, or a fusion protein comprising a portion ofCD80 linked to an Ig Fc sequence, under conditions wherein theinteraction of naturally-occurring B7-H1 with PD-1 or CD80 in saidmammal is reduced after said administering. The mammal can be a human.The elevated level of Bim can be based on Bim protein levels, or onBcl2l11 mRNA levels. The cancer can be a melanoma cancer, a breastcancer, a lung cancer, a renal cell carcinoma cancer, a pancreas cancer,a prostate cancer, a colon cancer, a brain cancer, a liver cancer, or anovarian cancer.

In another aspect, this document features a method for treating cancer,wherein said method comprises administering an anti-B7-H1 antibody, ananti-PD-1 antibody, an anti-CD80 antibody, a fusion protein comprising aportion of PD-1 linked to an immunoglobulin Fc sequence, or a fusionprotein comprising a portion of CD80 linked to an immunoglobulin Fcsequence to a mammal identified as containing an elevated level of Bim,wherein said antibody or fusion protein is administered under conditionswherein the interaction of naturally-occurring B7-H1 with PD-1 or CD80in said mammal is reduced after said administering. The mammal can be ahuman. The elevated level of Bim can be based on Bim protein levels, oron Bcl2l11 mRNA levels. The cancer can be a melanoma cancer, a breastcancer, a lung cancer, a renal cell carcinoma cancer, a pancreas cancer,a prostate cancer, a colon cancer, a brain cancer, a liver cancer, or anovarian cancer.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used to practicethe invention, suitable methods and materials are described below. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 contains a pair of graphs plotting the kinetics of CD8+ T-cellresponses to antigen stimulation. Wild type (WT) and B7-H1-deficient(KO) mice were immunized (i.p.) with OVA plus poly I:C, and K^(b)/OVAtetramer was used to identify antigen-specific CD8+ T cells in spleen(top panel) and liver (bottom panel) at the indicated times afterimmunization. Data show the percentage of tetramer+ CD8+ T cells(mean±SD of three mice per time point). One of two independentexperiments is shown. *p<0.05 compared with WT mice.

FIGS. 2A-2D contain a series of FACS scans and graphs showing enhancedmemory CD8+ T-cell population in the absence of B7-H1. Mice wereimmunized with OVA plus poly I:C, and were re-stimulated with OVA on day40 after immunization. On day 4 after re-stimulation, spleen cells wereisolated from naïve or immunized WT and B7-H1-deficient mice foranalysis. FIG. 2A, FACS scans showing the percentage of OVA-specifictetramer+ CD8+ T cells; *p<0.05 compared with WT mice. FIG. 2B graphplotting the absolute number of OVA-specific tetramer+ CD8+ T cells(mean±SD, n=3). FIG. 2C, FACS analysis of intracellular production ofcytokines in CD8+ T cells from immunized mice (mean±SD, n=3). FIG. 2D,graphs plotting in vivo cytolytic activity in immunized mice.OVA-peptide or control-peptide pulsed target cells (syngeneicsplenocytes) were labeled with high or low dose CFSE (5 μM forOVA-peptide pulsed cells; 0.5 μM for control-peptide pulsed cells) andmixed (1:1, 2.5×106 of each) and injected i.v. into WT orB7-H1-deficient mice. Histogram plots (left) show the percentage ofremaining target cells in the spleen 4 hours after target cell transfer.Bar graph (right) shows percentage of specific lysis in the spleen(mean±SD, n=3).

FIGS. 3A and 3B contain FACS scans and graphs showing enhanced memoryCD8+ T-cell recall responses and improved antitumor immunity in the lungin the absence of B7-H1. On day 35 after immunization, immunized ornaïve WT and B7-H1-deficient mice were injected (i.v.) with 5×10⁵B16-OVA tumor cells. FIG. 3A, percentage and absolute numbers of IFNγ+CD8+ T cells in the lung of immunized mice (mean±SD, n=3) on day 4 aftertumor injection. *p<0.01 compared with WT mice. FIG. 3B, metastatictumor foci in the lung tissue were identified and counted on day 20after tumor injection (mean±SD, n=5). N.S.: not significant.

FIGS. 4A-4D contain a series of FACS scans and a graph showing thatCD11a^(high) CD8+ T cells represent antigen-primed effector T cells.Spleen cells from naïve or immunized WT and B7-H1-deficient mice wereanalyzed by co-staining with anti-C D11a and K^(b)/OVA tetramer orfunctional markers. FIG. 4A, FACS scans showing the percentage ofCD11a^(high) CD8+ T cells from WT and B7-H1-deficient immunized mice.FIG. 4B, graph plotting average percentage of CD11a^(high) CD8+ T cellsfrom WT and B7-H1-deficient immunized mice (mean±SD, n=4). FIG. 4C, FACSscans showing the percentage of antigen-specific tetramer+(K^(b)/OVA-tet) cells in CD11a^(high) and CD11a^(low) CD8+ T-cellpopulation. FIG. 4D, FACS scans showing CTL functional assay ofCD11a^(high) and CD11a^(low) CD8+ T cells after a brief re-stimulationin vitro. Degranulation of CTLs was analyzed by CD107a mobilization,followed by intracellular staining for IFNγ. Numbers indicatepercentages of gated areas.

FIGS. 5A-5D contain a series of FACS scans and a graph showing fewerapoptotic antigen-primed CD8+ T cells in B7-H1-deficient mice. On day 7after immunization, spleen cells were analyzed for proliferation andapoptosis. FIGS. 5A and 5B, FACS scans showing Ki67 expression and BrdUincorporation, respectively, analyzed in CD11a^(high) or CD11a^(low)CD8+ T cells. Numbers are percentages of gated area in total CD8+ Tcells. FIG. 5C, FACS scans of TMRE^(low) Annexin V+ apoptotic cellsmeasured in CD11a^(high) and CD11a^(low) CD8+ T cells. FIG. 5D, graphplotting the percentage of apoptotic cells (TMRE^(low) Annexin V+) inCD11a^(high) CD8+ T cells (mean±SD, n=4).

FIGS. 6A-6D contain a series of histograms and a graph showing lower Bimlevels in antigen-primed CD8+ T cells in B7-H1-deficient mice. FIG. 6A,flow cytometry assay of the intracellular expression of Bim, Bcl-2 andBcl-xL in gated CD11a^(high) CD8+ T cells in the spleen of WT (red) andB7-H1-deficient (blue) mice on day 7 after immunization. Numbers aremean fluorescence intensity (MFI) of Bim expression. FIG. 6B, graphshowing average MFI of Bim expressed by CD11a^(high) CD8+ T cells(mean±SD, n=9). FIG. 6C, intracellular expression of Bim in CD11a^(high)CD8+ T cells in the liver of immunized mice. Numbers are MFI. FIG. 6D,Bim expression in total CD8+ T cells in the spleen of naive WT (red) andB7-H1-deficient (blue) mice.

FIG. 7 contains a series of histograms showing the extrinsic role ofB7-H1 in regulation of Bim. WT OT-1 CD8+ T cells (Thy1.1+) weretransferred in WT (red) or B7-H1-deficient (blue) host mice one daybefore immunization with OVA plus poly I:C. On day 7 after immunization,the OT-1 CD8+ T cells in the spleen and liver were identified by theThy1.1 marker and analyzed for intracellular expression of Bim. Numbersare MFI.

FIGS. 8A-8F show that B7-H1 co-stimulation induces upregulation of Bimprotein levels in activated T cells. Pre-activated CD8+ T cells wereincubated with platebound B7-H1 or control fusion protein (Fc) for 48hours in the presence of anti-CD3. FIG. 8A, Western blot showing Bimisoform expression in CD8+ T cells. FIG. 8B, histogram showingexpression of total Bim in CD8+ T cells co-stimulated with B7-H1 (blue)or control protein (red). Numbers are MFI. FIG. 8C, graph plottingaverage MFI of Bim expressed by activated CD8+ T cells (mean±SD, n=5).FIG. 8D, graph plotting the percentage of live (trypan blue exclusive)CD8+ T cells in culture (mean±SD, n=5). FIG. 8E, FACS scans indicatingapoptosis of CD8+ T cells isolated from WT, Bim-deficient, and Bcl-2transgenic (Tg) mice. Numbers show percentage of TMRE^(low) Annexin V+apoptotic T cells in total CD8+ T cells. FIG. 8F, graph plotting averageMFI of Bim expressed by CD8+ T cells in culture with anti-B7-H1 Ab(10B5, blocking B7-H1 binding to both PD-1 and CD80; 43H12, blockingB7-H1 binding to CD80 only), anti-PD-1 Ab (G4), or control Ab (10 μg/mLof each) (mean±SD, n=3).

FIGS. 9A-9C contain a series of plots showing that B7-H1 co-stimulationinhibits activation of Akt. Pre-activated CD8+ T cells were stimulatedwith plate-bound B7-H1 or control fusion protein (Fc). After 24 hours ofstimulation, CD8+ T cells were harvested and used for analysis. FIG. 9A,graph plotting analysis of Bcl2l11 transcript levels by real-time qPCRusing the comparative CT method. GAPDH served as the internal controlgene. Graph shows fold change (mean±SD, n=4). FIG. 9B, histogramsplotting phosphorylation of Akt (left) and mTOR (right), analyzed byintracellular staining of CD8+ T cells with anti-phospho-Akt andanti-phospho-mTOR Abs. Numbers show percentage of positive stainedcells. FIG. 9C, bar graph plotting average MFI of phospho-Akt andphospho-mTOR expression (mean±SD, n=3). N.S.: not significant.

FIG. 10 contains representative nucleic acid (top) and amino acid(bottom) sequences for human B7-H1 (SEQ ID NOS:1 and 2, respectively).

FIG. 11 contains representative nucleic acid (top) and amino acid(bottom) sequences for human PD-1 (SEQ ID NOS:3 and 4, respectively).

FIGS. 12A and 12B contain representative nucleic acid (12A) and aminoacid (12B) sequences for human CD80 (SEQ ID NOS:5 and 6, respectively).

FIG. 13 contains a plot (left) showing the identification of CD8+ Tcells based on their expression of CD11a^(high) and PD-1+ (left panel).Lymphocytes were stained with CD8, CD11a and PD-1, followed byintracellular staining for Bim. FIG. 13 also contains a histogramplotting expression of Bim by subsets of CD8+ T cells (Tn: T naïvecells; PD-1−, PD-1 negative primed cells; PD-1+, PD-1 positive primedcells). Only the PD-1+ primed cells CD8+ T cells expressed high levelsof Bim.

FIG. 14 is a pair of graphs plotting the level of Bim expression intumor-reactive PD-1+ CD11a^(high) CD8+ T cells in the peripheral bloodof 26 melanoma patients as compared to 11 normal, healthy controls (leftpanel, P=0.007 by unpaired Student T test), and in tumor-reactive PD-1+CD11a^(high) CD8+ T cells in the peripheral blood of 11 prostate cancerpatients as compared to 11 normal, healthy controls (right panel,P=0.001 by unpaired Student T test).

FIG. 15 is a pair of graphs plotting Bim expression in PD-1 negative(PD-1−) and PD-1 positive (PD-1+) CD11a^(high) CD8+ T cells frommelanoma patients (left) and healthy controls (right). Bim wassignificantly increased in the PD-1+ populations (p=0.0081) in melanomapatients.

FIG. 16 is a pair of graphs plotting the level of Bim expression intumor-reactive PD-1+ CD11a^(high) CD8+ T cells in the peripheral bloodof 26 melanoma patients as compared to 11 normal, healthy controls (leftpanel, indicating “Bim low” and “Bim high” samples), and plotting thesurvival rate for “Bim low” vs. “Bim high” patients (right panel).

FIG. 17A is a pair of graphs showing that B7-H1 protein inducedexpression of Bim in human pre-activated CD8+ T cells. FIG. 17B is apicture of a Western blot showing Bim levels in the cells.

FIG. 18 is a pair of graphs showing that an anti-PD-1 antibodysignificantly blocked B7-H1-induced Bim up-regulation in a dosedependent fashion (left panel), and that the blocking effects of theanti-PD-1 antibody were inversely correlated with the higher levels ofBim induced by B7-H1 (right panel).

FIG. 19 is a pair of graphs showing that the frequency of Bim+PD-1+ CD8T cells was significantly higher in the peripheral blood of melanomapatients before treatment than in a healthy control group (left panel),and that after anti-PD-1 antibody therapy, about 67% of the melanomapatients demonstrated a significant reduction in the frequency ofBim+PD-1+ CD8 T cells (right panel).

FIG. 20 is a pair of graphs showing that B7-H1 expressed by tumor cellsinduced Bim up-regulation in human pre-activated CD8 T cells.

FIG. 21 is a pair of photographs (left) and a graph (right) showing thatBim expression was associated with B7-H1 expression in human renal cellcarcinoma (RCC). In particular, the graph in the right panel shows thatBim+ tumor infiltrating lymphocytes (TILs) were increased in B7-H1positive tumor tissues. Bim reactivity scores: 0, absence; 1, focal; 2,moderate; 3, marked. Contingency analysis using Fisher's exact test(p<0.01).

FIG. 22 is a pair of graphs showing that Bim expression was correlatedwith Granzyme B and T-bet (a transcription factor of effector T cells)expressed by cancer-related PD-1+ CD11a^(high) CD8+ T cells, suggestingthat Bim expression is associated with effector CD8+ T celldifferentiation.

FIG. 23 is a pair of graphs showing that Bim expression declined inPD-1+ CD11a^(high) CD8 T cells following radiotherapy in some melanoma(left panel) and prostate (right panel) cancer patients.

DETAILED DESCRIPTION

This document provides materials and methods for identifying patients asbeing more likely to benefit from checkpoint blockade therapy, materialsand methods for determining optimal therapeutic timing and dosingschedules, and methods and materials for treating cancer. For example,this document provides methods and materials for identifying a mammal(e.g., a human) as having an elevated level of Bim, and treating themammal with a molecule that can interfere with the interaction betweenB7-H1 and PD-1, and/or the interaction between B7-H1 and CD80 (e.g., anantibody against B7-H1, PD-1, or CD80, or with a fusion proteincontaining a portion of PD-1 or a portion of CD80 fused to animmunoglobulin (Ig) Fc domain). As described herein, elevated levels ofBim can be related to increased apoptosis of antigen-primed CD8+ Tcells, but inhibiting the interaction of B7-H1 with PD-1 or CD80 canlead to reduced levels of Bim and reduced T cell apoptosis.

The term “elevated level” as used herein with respect to a level of Bimrefers to a level that is greater (e.g., 50% greater, 2-fold greater,3-fold greater, or more than 3-fold greater) than a reference level ofBim. The term “reference level” as used herein with respect to Bimrefers to the level of Bim typically observed in healthy mammals withoutcancer. For example, a reference level of Bim can be the average levelof Bim present in samples obtained from a random sampling of 50 humansfree of cancer.

The presence of an elevated level of Bim can be determined by measuring,for example, protein levels or nucleic acid levels. For example, thelevel of Bim protein can be measured in a sample of blood (e.g., aperipheral blood sample) or another bodily fluid from a mammal withcancer or from a control mammal, using cell staining, western blotting,or other immunological techniques. The level of Bim expression also canbe measured at the nucleic acid level, using Northern blotting, or anyother method suitable for determining mRNA levels of Bcl2l11, whichencodes the Bim protein. In some cases, Bim protein or nucleic acidlevels can be measured in tumor tissue samples, ascites samples, orlymphoid organ samples. It will be appreciated that levels fromcomparable samples are used when determining whether or not a particularlevel is an elevated level.

A representative example of a human B7-H1 nucleic acid has the sequenceset forth in GENBANK® Accession No. AF177937 (GI No. 6708118) (SEQ IDNO:1; FIG. 10), and a representative human B7-H1 polypeptide has thesequence set forth in GENBANK® Accession No. AAF25807 (GI No. 6708119)(SEQ ID NO:2; FIG. 10). A representative example of a human PD-1 nucleicacid can have the sequence set forth in GENBANK® Accession No.BC074740.2 (GI No. 50960296) (SEQ ID NO:3; FIG. 11), and representativeexample of a human PD-1 polypeptide has the sequence set forth inGENBANK® Accession No. AAH74740.1 (GI No. 49902307) (SEQ ID NO:4; FIG.11).

A representative example of a human CD80 nucleic acid has the sequenceset forth in NCBI Reference No. NM_005191.3 (GI No. 113722122) (SEQ IDNO:5; FIG. 12A), and a representative example of a human CD80polypeptide has the sequence set forth in NCBI Reference No. NP_005182.1(GI No. 4885123) (SEQ ID NO:6; FIG. 12B).

Once the level of Bim within a sample from a mammal is determined, thelevel can be compared to a reference level and used to classify themammal as having or lacking an elevated level of Bim.

Once a mammal has been identified as having an elevated level of Bim asdescribed herein, the mammal can be administered a molecule thatinhibits the interaction between B7-H1 and PD-1 and/or the interactionbetween B7-H1 and CD80. Examples of such molecules include, withoutlimitation, antibodies (e.g., anti-B7-H1 antibodies, anti-PD-1antibodies, or anti-CD80 antibodies), and fusion proteins (e.g., PD-1fusion proteins or CD80 fusion proteins). Such fusion proteins cancontain, for example, the extracellular domain of PD-1 fused to an IgGFc domain, or the extracellular domain of CD80 fused to an IgG Fcdomain. After administration, the antibody/ies or fusion protein(s) canbind B7-H1, thus reducing or blocking B7-H1's action in inducing Bim upregulation.

The term “antibody” includes monoclonal antibodies, polyclonalantibodies, recombinant antibodies, humanized antibodies (Jones et al.(1986) Nature 321:522-525; Riechmann et al. (1988) Nature 332:323-329;and Presta (1992) Curr. Op. Struct. Biol. 2:593-596), chimericantibodies (Morrison et al. (1984) Proc. Natl. Acad. Sci. USA81:6851-6855), multispecific antibodies (e.g., bispecific antibodies)formed from at least two antibodies, and antibody fragments. The term“antibody fragment” comprises any portion of the afore-mentionedantibodies, such as their antigen binding or variable regions. Examplesof antibody fragments include Fab fragments, Fab′ fragments, F(ab′)2fragments, Fv fragments, diabodies (Hollinger et al. (1993) Proc. Natl.Acad. Sci. USA 90:6444-6448), single chain antibody molecules (Plückthunin: The Pharmacology of Monoclonal Antibodies 113, Rosenburg and Moore,eds., Springer Verlag, N.Y. (1994), 269-315) and other fragments as longas they exhibit the desired capability of binding to B7-H1, PD-1, orCD80.

Examples of anti-human B7-H1 antibodies include, without limitation,anti-human B7-H1 antibodies commercially available from Biolegend (e.g.,Catalog No. 329701 or 329702; San Diego, Calif.) or eBioscience (e.g.,Catalog No. 14-5983-80 or 14-5983-82).

Examples of anti-human PD-1 antibodies include, without limitation,anti-human PD-1 antibodies commercially available from Biolegend (e.g.,Catalog No. 329904 or 329905) or eBioscience (Catalog No. 12-2799-42;San Diego, Calif.).

Examples of anti-human CD80 antibodies include, without limitation,anti-human CD8 antibodies commercially available from Biolegend (e.g.,Catalog No. 305201 or 305202) or eBioscience (e.g., Catalog No.14-0809-80 or 14-0809-82).

The term “antibody,” as used herein, also includes antibody-likemolecules that contain engineered sub-domains of antibodies or naturallyoccurring antibody variants. These antibody-like molecules may besingle-domain antibodies such as V_(H)-only or V_(L)-only domainsderived either from natural sources such as camelids (Muyldermans et al.(2001) Rev. Mol. Biotechnol. 74:277-302) or through in vitro display oflibraries from humans, camelids or other species (Holt et al. (2003)Trends Biotechnol. 21:484-90). In certain embodiments, the polypeptidestructure of the antigen binding proteins can be based on antibodies,including, but not limited to, minibodies, synthetic antibodies(sometimes referred to as “antibody mimetics”), human antibodies,antibody fusions (sometimes referred to as “antibody conjugates”), andfragments thereof, respectively.

An “Fv fragment” is the minimum antibody fragment that contains acomplete antigen-recognition and -binding site. This region consists ofa dimer of one heavy chain variable domain and one light chain variabledomain in tight, non-covalent association. It is in this configurationthat the three CDR's of each variable domain interact to define anantigen-binding site on the surface of the VH-VL dimer. Collectively,the six CDR's confer antigen-binding specificity to the antibody.However, even a single variable domain (or half of an Fv comprising onlythree CDR's specific for an antigen) has the ability to recognize andbind the antigen, although usually at a lower affinity than the entirebinding site. The “Fab fragment” also contains the constant domain ofthe light chain and the first constant domain (C_(H)1) of the heavychain. The “Fab fragment” differs from the “Fab′ fragment” by theaddition of a few residues at the carboxy terminus of the heavy chainC_(H)1 domain, including one or more cysteines from the antibody hingeregion. The “F(ab′)2 fragment” originally is produced as a pair of “Fab′fragments” which have hinge cysteines between them. Methods of preparingsuch antibody fragments, such as papain or pepsin digestion, are knownto those skilled in the art.

An antibody can be of the IgA-, IgD-, IgE, IgG- or IgM-type, includingIgG- or IgM-types such as, without limitation, IgG1-, IgG2-, IgG3-,IgG4-, IgM1- and IgM2-types. For example, in some cases, the antibody isof the IgG1-, IgG2- or IgG4-type.

In some embodiments, antibodies as used in the methods described hereincan be fully human or humanized antibodies. Human antibodies can avoidcertain problems associated with xenogeneic antibodies, such asantibodies that possess murine or rat variable and/or constant regions.First, because the effector portion is human, it can interact betterwith other parts of the human immune system, e.g., to destroy targetcells more efficiently by complement-dependent cytotoxicity orantibody-dependent cellular cytotoxicity. Second, the human immunesystem should not recognize the antibody as foreign. Third, half-life inhuman circulation will be similar to naturally occurring humanantibodies, allowing smaller and less frequent doses to be given.Methods for preparing human antibodies are known in the art.

In addition to human antibodies, “humanized” antibodies can have manyadvantages. Humanized antibodies generally are chimeric or mutantmonoclonal antibodies from mouse, rat, hamster, rabbit or other species,bearing human constant and/or variable region domains or specificchanges. Techniques for generating humanized antibodies are well knownto those of skill in the art. For example, controlled rearrangement ofantibody domains joined through protein disulfide bonds to form new,artificial protein molecules or “chimeric” antibodies can be utilized(Konieczny et al. (1981) Haematologia (Budap.) 14:95). Recombinant DNAtechnology can be used to construct gene fusions between DNA sequencesencoding mouse antibody variable light and heavy chain domains and humanantibody light and heavy chain constant domains (Morrison et al. (1984)Proc. Natl. Acad. Sci. USA 81:6851).

DNA sequences encoding antigen binding portions or complementaritydetermining regions (CDR's) of murine monoclonal antibodies can begrafted by molecular means into DNA sequences encoding frameworks ofhuman antibody heavy and light chains (Jones et al. (1986) Nature321:522; Riechmann et al. (1988) Nature 332:323). Expressed recombinantproducts are called “reshaped” or humanized antibodies, and comprise theframework of a human antibody light or heavy chain and antigenrecognition portions, CDR's, of a murine monoclonal antibody.

Other methods for designing heavy and light chains and for producinghumanized antibodies are described in, for example, U.S. Pat. Nos.5,530,101; 5,565,332; 5,585,089; 5,639,641; 5,693,761; 5,693,762; and5,733,743. Yet additional methods for humanizing antibodies aredescribed in U.S. Pat. Nos. 4,816,567; 4,935,496; 5,502,167; 5,558,864;5,693,493; 5,698,417; 5,705,154; 5,750,078; and 5,770,403, for example.

Molecules that interfere with the interaction between B7-H1 and PD-1,and/or the interaction between B7-H1 and CD80, as described herein(e.g., antibodies against B7-H1, PD-1, and CD80, as well as fusionproteins containing portions of PD-1 or CD80 linked to an Ig Fc domain),can be incorporated into pharmaceutical compositions for treatment ofcancer. Thus, this document also provides the use of such molecules inthe manufacture of medicaments for treating cancer. The compositions canfurther include one or more pharmaceutically acceptable carriers,diluents and/or adjuvants. The potency of the pharmaceuticalcompositions provided herein typically is based on the binding of theantibody or fusion protein to B7-H1.

A “pharmaceutically acceptable carrier” (also referred to as an“excipient” or a “carrier”) is a pharmaceutically acceptable solvent,suspending agent, stabilizing agent, or any other pharmacologicallyinert vehicle for delivering one or more therapeutic compounds to asubject, which is nontoxic to the cell or mammal being exposed theretoat the dosages and concentrations employed. Pharmaceutically acceptablecarriers can be liquid or solid, and can be selected with the plannedmanner of administration in mind so as to provide for the desired bulk,consistency, and other pertinent transport and chemical properties, whencombined with one or more of therapeutic compounds and any othercomponents of a given pharmaceutical composition. Typicalpharmaceutically acceptable carriers that do not deleteriously reactwith amino acids include, by way of example and not limitation: water,saline solution, binding agents (e.g., polyvinylpyrrolidone orhydroxypropyl methylcellulose), fillers (e.g., lactose and other sugars,gelatin, or calcium sulfate), lubricants (e.g., starch, polyethyleneglycol, or sodium acetate), disintegrates (e.g., starch or sodium starchglycolate), and wetting agents (e.g., sodium lauryl sulfate).Pharmaceutically acceptable carriers also include aqueous pH bufferedsolutions or liposomes (small vesicles composed of various types oflipids, phospholipids and/or surfactants which are useful for deliveryof a drug to a mammal). Further examples of pharmaceutically acceptablecarriers include buffers such as phosphate, citrate, and other organicacids, antioxidants such as ascorbic acid, low molecular weight (lessthan about 10 residues) polypeptides, proteins such as serum albumin,gelatin, or immunoglobulins, hydrophilic polymers such aspolyvinylpyrrolidone, amino acids such as glycine, glutamine,asparagine, arginine or lysine, monosaccharides, disaccharides, andother carbohydrates including glucose, mannose or dextrins, chelatingagents such as EDTA, sugar alcohols such as mannitol or sorbitol,salt-forming counterions such as sodium, and/or nonionic surfactantssuch as TWEEN™, polyethylene glycol (PEG), and PLURONICS™.

Pharmaceutical compositions can be formulated by mixing one or moreactive agents with one or more physiologically acceptable carriers,diluents, and/or adjuvants, and optionally other agents that are usuallyincorporated into formulations to provide improved transfer, delivery,tolerance, and the like. A pharmaceutical composition can be formulated,e.g., in lyophilized formulations, aqueous solutions, dispersions, orsolid preparations, such as tablets, dragees or capsules. A multitude ofappropriate formulations can be found in the formulary known to allpharmaceutical chemists: Remington's Pharmaceutical Sciences (18th ed,Mack Publishing Company, Easton, Pa. (1990)), particularly Chapter 87 byBlock, Lawrence, therein. These formulations include, for example,powders, pastes, ointments, jellies, waxes, oils, lipids, lipid(cationic or anionic) containing vesicles (such as LIPOFECTIN™), DNAconjugates, anhydrous absorption pastes, oil-in-water and water-in-oilemulsions, emulsions carbowax (polyethylene glycols of various molecularweights), semi-solid gels, and semi-solid mixtures containing carbowax.Any of the foregoing mixtures may be appropriate in treatments andtherapies as described herein, provided that the active agent in theformulation is not inactivated by the formulation and the formulation isphysiologically compatible and tolerable with the route ofadministration. See, also, Baldrick (2000) Regul. Toxicol. Pharmacol.32:210-218; Wang (2000) Int. J. Pharm. 203:1-60; Charman (2000) J.Pharm. Sci. 89:967-978; and Powell et al. (1998) PDA J. Pharm. Sci.Technol. 52:238-311), and the citations therein for additionalinformation related to formulations, excipients and carriers well knownto pharmaceutical chemists.

Pharmaceutical compositions include, without limitation, solutions,emulsions, aqueous suspensions, and liposome-containing formulations.These compositions can be generated from a variety of components thatinclude, for example, preformed liquids, self-emulsifying solids andself-emulsifying semisolids. Emulsions are often biphasic systemscomprising of two immiscible liquid phases intimately mixed anddispersed with each other; in general, emulsions are either of thewater-in-oil (w/o) or oil-in-water (o/w) variety. Emulsion formulationshave been widely used for oral delivery of therapeutics due to theirease of formulation and efficacy of solubilization, absorption, andbioavailability.

Compositions and formulations can include sterile aqueous solutions,which also can contain buffers, diluents and other suitable additives(e.g., penetration enhancers, carrier compounds and otherpharmaceutically acceptable carriers). Compositions additionally cancontain other adjunct components conventionally found in pharmaceuticalcompositions. Thus, the compositions also can include compatible,pharmaceutically active materials such as, for example, antipruritics,astringents, local anesthetics or anti-inflammatory agents, oradditional materials useful in physically formulating various dosageforms of the compositions provided herein, such as dyes, flavoringagents, preservatives, antioxidants, opacifiers, thickening agents andstabilizers. Furthermore, the composition can be mixed with auxiliaryagents, e.g., lubricants, preservatives, stabilizers, wetting agents,emulsifiers, salts for influencing osmotic pressure, buffers, colorings,flavorings, and aromatic substances. When added, however, such materialsshould not unduly interfere with the biological activities of thepolypeptide components within the compositions provided herein. Theformulations can be sterilized if desired.

In some embodiments, a composition containing an antibody or fusionprotein as provided herein (e.g., an anti-B7-H7, anti-PD-1, or anti-CD80antibody, or a PD-1 FC or CD80 Fc fusion protein) can be in the form ofa solution or powder with or without a diluent to make an injectablesuspension. The composition may contain additional ingredientsincluding, without limitation, pharmaceutically acceptable vehicles,such as saline, water, lactic acid, mannitol, or combinations thereof,for example.

Any appropriate method can be used to administer a molecule as describedherein to a mammal. Administration can be, for example, parenteral(e.g., by subcutaneous, intrathecal, intraventricular, intramuscular, orintraperitoneal injection, or by intravenous drip). Administration canbe rapid (e.g., by injection) or can occur over a period of time (e.g.,by slow infusion or administration of slow release formulations). Insome embodiments, administration can be topical (e.g., transdermal,sublingual, ophthalmic, or intranasal), pulmonary (e.g., by inhalationor insufflation of powders or aerosols), or oral. In addition, acomposition containing an antibody or fusion protein as described hereincan be administered prior to, after, or in lieu of surgical resection ofa tumor.

A composition containing an antibody (e.g., an anti-B7-H1 antibody,anti-PD-1 antibody, or anti-CD80 antibody) or a fusion protein (e.g., aPD-1 Fc fusion or a CD80 Fc fusion) can be administered to a mammal inany appropriate amount, at any appropriate frequency, and for anyappropriate duration effective to achieve a desired outcome (e.g., toincrease progression-free survival). In some cases, a compositioncontaining an antibody or fusion protein as described herein can beadministered to a mammal having cancer to reduce the progression rate ofthe cancer by 5, 10, 25, 50, 75, 100, or more percent. For example, theprogression rate can be reduced such that no additional cancerprogression is detected. Any appropriate method can be used to determinewhether or not the progression rate of cancer is reduced. For skincancer (e.g., melanoma), for example, the progression rate can beassessed by imaging tissue at different time points and determining theamount of cancer cells present. The amounts of cancer cells determinedwithin tissue at different times can be compared to determine theprogression rate. After treatment as described herein, the progressionrate can be determined again over another time interval. In some cases,the stage of cancer after treatment can be determined and compared tothe stage before treatment to determine whether or not the progressionrate has been reduced.

In some cases, a composition containing an antibody or a fusion proteinas described herein can be administered to a mammal having cancer underconditions where progression-free survival is increased (e.g., by 5, 10,25, 50, 75, 100, or more percent) as compared to the medianprogression-free survival of corresponding mammals having untreatedcancer or the median progression-free survival of corresponding mammalshaving cancer and treated with other therapies (e.g., chemotherapeuticagents). Progression-free survival can be measured over any length oftime (e.g., one month, two months, three months, four months, fivemonths, six months, or longer).

Administration to a mammal of a molecule as set forth herein can resultin increased numbers of naturally-occurring tumor-reactive CD8+ T cells,which can exert anti-cancer effects against cancer cells present withinthe mammal.

An effective amount of a composition containing a molecule as providedherein can be any amount that reduces the progression rate of cancer,increases the progression-free survival rate, or increases the mediantime to progression without producing significant toxicity to themammal. Optimum dosages can vary depending on the relative potency ofindividual polypeptides (e.g., antibodies and fusion proteins), and cangenerally be estimated based on EC₅₀ found to be effective in in vitroand in vivo animal models. Typically, dosage is from 0.01 μg to 100 gper kg of body weight. For example, an effective amount of an antibodyor fusion protein can be from about 1 mg/kg to about 100 mg/kg (e.g.,about 5 mg/kg, about 10 mg/kg, about 20 mg/kg, about 50 mg/kg, or about75 mg/kg). If a particular mammal fails to respond to a particularamount, then the amount of the antibody or fusion protein can beincreased by, for example, two fold. After receiving this higherconcentration, the mammal can be monitored for both responsiveness tothe treatment and toxicity symptoms, and adjustments made accordingly.The effective amount can remain constant or can be adjusted as a slidingscale or variable dose depending on the mammal's response to treatment.Various factors can influence the actual effective amount used for aparticular application. For example, the frequency of administration,duration of treatment, use of multiple treatment agents, route ofadministration, and severity of the cancer may require an increase ordecrease in the actual effective amount administered.

The frequency of administration can be any frequency that reduces theprogression rate of cancer, increases the progression-free survivalrate, or increases the median time to progression without producingsignificant toxicity to the mammal. For example, the frequency ofadministration can be once or more daily, biweekly, weekly, monthly, oreven less. The frequency of administration can remain constant or can bevariable during the duration of treatment. A course of treatment caninclude rest periods. For example, a composition containing an antibodyor fusion protein as provided herein can be administered over a two weekperiod followed by a two week rest period, and such a regimen can berepeated multiple times. As with the effective amount, various factorscan influence the actual frequency of administration used for aparticular application. For example, the effective amount, duration oftreatment, use of multiple treatment agents, route of administration,and severity of the cancer may require an increase or decrease inadministration frequency.

An effective duration for administering a composition provided hereincan be any duration that reduces the progression rate of cancer,increases the progression-free survival rate, or increases the mediantime to progression without producing significant toxicity to themammal. Thus, the effective duration can vary from several days toseveral weeks, months, or years. In general, the effective duration forthe treatment of cancer can range in duration from several weeks toseveral months. In some cases, an effective duration can be for as longas an individual mammal is alive. Multiple factors can influence theactual effective duration used for a particular treatment. For example,an effective duration can vary with the frequency of administration,effective amount, use of multiple treatment agents, route ofadministration, and severity of the cancer.

After administering a composition provided herein to a mammal, themammal can be monitored to determine whether or not the cancer wastreated. For example, a mammal can be assessed after treatment todetermine whether or not the progression rate of the cancer has beenreduced (e.g., stopped). Any method, including those that are standardin the art, can be used to assess progression and survival rates.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES Example 1—Materials and Methods

Mice, Cell Lines and Reagents:

Female CD45.2+ C57BL/6 mice were purchased from Taconic Farms andCD45.1+ congenic C57BL/6-Ly5.1 mice were purchased from National CancerInstitute. OT-1 TCR (Thy 1.1+) transgenic mice were provided by T. Tian(Harvard University, Boston, Mass.). B7-H1-deficient C57BL/6 mice wereprovided by L. Chen (Yale University, New Haven, Conn.; Dong et al.,Immunity 20:327-336, 2004). Bcl2l11−/− mice and Cd80−/− mice werepurchased from Jackson Laboratory. Cd80−/− mice were crossbred into WTOT-1 mice and produced Cd80−/− OT-1 mice. Bcl-2 transgenic mice wereprovided by V. Shapiro (Mayo Clinic, Rochester). Mice were maintainedunder pathogen-free conditions and used at 8-12 weeks of age. B16-OVAmurine melanoma cells were provided by R. Vile (Mayo Clinic, Rochester,Minn.), and were cultured in RPMI 1640 medium (Cellgro) with 10% FBS(Life Technologies), 1 U/mL penicillin, 1 μg/mL streptomycin and 20 mMHEPES buffer (all from Mediatech). Hamster anti-mouse B7-H1 mAb (10B5)and PD-1 (G4) was obtained from hybridoma cells provided by L. Chen.Hamster anti-mouse B7-H1 mAb (43H12) was provided by K. Tamada (JohnHopkins University).

Flow Cytometry Analysis:

Class I MEW (KbOVA peptide SIINFEKL; SEQ ID NO:1) tetramer and negativecontrol tetramer were purchased from Beckman Coulter.Fluorochrome-conjugated Abs against CD8, CD11a, Fas (CD95), Fas ligand,CD90.1 (Thy 1.1), CD90.2 (Thy 1.2), CD107a, IFNγ, IL-2 and TNFα werepurchased from BD Biosciences, BioLegend, or eBiosciences. To detectintracellular cytokine levels, cells were incubated with GolgiPlug (BDBiosciences) for 4 hours prior to analysis. Cells were stained forsurface antigens, and then incubated in Fixation Buffer (BioLegend) for20 minutes at room temperature, followed by permeabilization inPermeabilization Wash Buffer (BioLegend). Fixed and permeabilized cellswere then stained with Abs for 20 minutes at room temperature. Abs toAkt, Bcl-xL, Bcl-2, Bim and mTOR and fluorochrome-conjugated secondaryAbs were purchased from Cell Signaling (Danvers, Mass.). To detectintracellular levels of Akt, Bcl-xL, Bcl-2, Bim and mTOR, T cells werefirst stained for surface antigens, then fixed with 2% paraformaldehydefor 10 minutes at 37° C., followed by permeabilization with ice-coldmethanol for 30 minutes. After blocking with 15% rat serum for 15minutes, cells were stained with Abs for 1 hour at room temperature.After staining, cells were washed three times with incubation bufferbefore analysis. At least 100,000 viable cells were live gated onFACScan or FACSCailbur (BD Biosciences) instrumentation. Flow cytometryanalysis was performed using FlowJo software (Tree Star).

T-Cell Immunization, Activation, Apoptosis Assay and ProliferationAssay:

Mice were immunized by i.p. injection of 0.5 mg ovalbumin (OVA, fromSigma-Aldrich) and 50 μg poly (I:C) (Sigma Aldrich). For in vitro T-cellactivation and apoptosis assay, purified CD8+ T cells were labeled withCFSE (Invitrogen-Molecular Probes) and incubated with OVA peptide₂₅₇₋₂₆₄(Mayo Clinic Core Facilities) at 0.2 μg/mL for 72 hours. Apoptosis ofCD8+ T cells was analyzed by staining using Annexin V (BD Biosciences)and TMRE (tetramethylrhodamine ethyl ester, Invitrogen/Molecular ProbesT-669). Proliferation was also measured by detection of BrdUincorporation and Ki67 staining. Immunized mice were injected i.p. with0.8 mg/mL BrdU (BD Biosciences) on days 4 through 6 followingimmunization. On day 7 after immunization BrdU incorporation wasdetermined by intra-nuclear staining with anti-BrdU (B9285,Sigma-Aldrich) and anti-Ki67 (556027, BD Biosciences).

In Vivo CTL Assay.

For the in vivo CTL assay, OVA₂₅₇₋₂₆₄ peptide-pulsed or controlpeptide-pulsed spleen cells (as target cells) from syngeneic mice werelabeled with a high dose of CFSE (5 μM) or low dose of CFSE (0.5 μM),mixed at 1:1 (2.5×10⁶ of each) before injection. Target cells were i.v.injected into immunized mice on day 4 after re-challenge with cognateantigen protein. The CTL activity was determined 4 hours after targetcell transfer. Specific lysis is calculated using the followingformulas: ratio=(% CFSEhigh/% CFSElow), % specific lysis=[1−(ratioprimed/ratio unprimed)]×100%.

Tumor Studies:

Mice were inoculated i.v. with 5×10⁵ B16-OVA tumor cells on day 25 afterimmunization. On day 21-post tumor injection, mice were sacrificed andthe lung tissue was perfused with PBS. The number of tumor foci on thelung tissue was counted.

T-Cell Transfer Experiments:

Purified CD8+ T cells (1×10⁶) from Thy1.1+ OT-1 transgenic mice werei.v. injected into Thy 1.2+ WT or B7-H1-deficient recipient mice,followed by immunization with OVA plus poly I:C. On day 7 afterimmunization, transferred CD8+ T cells were identified by theirexpression of Thy1.1 and used for detection of intracellular expressionof Bim, Bcl-2 and Bcl-xL. Equal numbers of Cd80−/− (CD45.2+) and WT OT-1(Thy1.1+, CD45.2+) CD8+ T cells (10⁶ of each) were i.v. injected intoCD45.1+ mice followed with immunization of OVA and poly I:C. Thetransferred OT-1 CD8+ T cells in the spleen were identified by flowcytometry.

In Vitro T-Cell Activation and Culturing with Fusion Proteins:

Spleen cells were harvested from naïve mice and pre-activated with ConA(5 μg/mL, L7647, Sigma-Aldrich) for 48 hours. Following activation, CD8+T cells were purified (EasySep CD8+ T-cell negative selection kit, StemCell Technologies) and incubated with plate-bound anti-CD3 (BDBiosciences) and B7-H1 Fc fusion protein or control Fc protein (R&DSystems). Cultures were maintained for indicated time periods, and thencells were harvested for analysis.

Western Blotting:

Cells were lysed with NETN buffer (0.5% NP40, 150 mM NaCl, 50 mM Trisand 1 mM EDTA). Cell lysates were boiled and run on SDS-PAGE gels(BioRad), transferred to nitrocellulose membrane (Millipore), andblotted using standard procedures.

Quantitative RT-PCR:

Total RNA was isolated from purified CD8+ T cells (RNeasy Kit, Qiagen),and reverse transcribed (iScriptcDNA synthesis kit, BioRad). Sampleswere analyzed for Bim transcript levels using Bcl2l11 primers (Qiagen)and QuantiFast SYBR Green PCR Master Mix (Qiagen) on an iCycler(BioRad). GAPDH levels were used to normalize data by the comparative CTmethod.

Statistical Analysis:

A two-sided, unpaired or paired Student's t-test was used to assessstatistical differences in experimental groups. A p value <0.05 wasconsidered statistically significant.

Example 2—More Memory T Cells are Generated in the Absence of B7-H1

The kinetics of CD8+ T-cell responses in the spleen and liver of WT andB7-H1-deficient C57BL/6 mice were compared following immunization withovalbumin (OVA) protein and polyinosinic:polycytidylic acid (poly (I:C))as adjuvant. An increased number of CD8+ T cells was observed at thepeak of the immune response (day 7-post immunization) in the spleen andliver of B7-H1-deficient mice as compared with WT mice. During thecontraction phase (days 7 to 14 post-immunization), there was asignificant delay in the reduction of antigen-specific CD8+ T cells inthe spleen and liver of B7-H1-deficient mice as compared with WT mice.On day 40 following immunization, more antigen-specific memory CD8+ Tcells were detected in B7-H1-deficient mice as compared with WT mice(FIG. 1). These data suggested that host B7-H1 may regulate the extentof expansion and contraction of effector CD8+ T cells, thus influencingthe size of the memory CD8+ T-cell pool in both lymphoid andnon-lymphoid tissues.

Studies were conducted to examine the extent to which B7-H1 regulatesthe generation of memory CD8+ T cells in immunized mice, usingK^(b)OVA₂₅₇₋₂₆₄ tetramer (K^(b)OVA-tet) to detect antigen-primed memoryCD8+ T cells in the spleen on day 4 after in vivo restimulation (OVAprotein, administered on day 40 after primary immunization). Day 4 wasselected for analysis because at this time point it is possible todistinguish a recall response from the primary response (which takes 7days to establish). Thus, naïve mice did not show a significant increaseof antigen-specific CD8+ T cells on day 4 after immunization (FIG. 2A).The frequency of K^(b)OVA-tet+ CD8+ T cells increased more than 2-foldin immunized B7-H1-deficient mice (0.38%) as compared with WT mice(0.16%; p<0.05; FIG. 2A). This increase was reflected in the absolutecell numbers (p=0.001; FIG. 2B). In addition to having increased numbersof memory CD8+ T cells, an increased percentage of memory CD8+ T cellscapable of producing multiple cytokines was detected in the spleens ofB7-H1-deficient mice (0.73% IFNγ+/TNFα+, 0.17% IFNγ+/IL-2+) as comparedwith WT mice (0.24% IFNγ+/TNFα+, 0.07% IFNγ+/IL-2+; p<0.05; FIG. 2C). Anin vivo CTL assay to measure cytolytic activity of the memory CD8+ Tcells also was performed. On day 4 after in vivo re-stimulation, OVApeptide- or control peptide-pulsed target cells (syngeneic splenocyteslabeled with either high or low CF SE) were injected into immunized WTand B7-H1-deficient mice. Four hours following cell injection, theremaining CFSE positive cells in the spleen were analyzed. Memory CD8+ Tcells in the B7-H1-deficient mice lysed more OVA-peptide pulsed targetcells (33.5%) than those in WT mice (9.3%, p<0.01; FIG. 2D).Collectively, these data suggest that B7-H1 negatively regulates thegeneration of memory CD8+ T cells in immunized mice.

A hallmark of memory CD8+ T cells is their rapid recall response tocognate antigens, so studies were conducted to determine whether theincreased memory pool in B7-H1-deficient mice would lead to a moreprotective recall response. B16-OVA melanoma tumor cells (engineered toexpress OVA) were injected into immunized WT and B7-H1-deficient mice.Intravenously injected B16-OVA tumor cells form metastases in the lung,and antitumor immunity can be monitored by counting the number of tumorfoci. On day 4 following intravenous injection of 5×10⁵ B16-OVA tumorcells, the frequency of functional memory CD8+ T cells in the lungs ofWT and B7-H1-deficient mice was determined by intracellular staining forIFNγ. About 4 to 5-fold more IFNγ+ CD8+ T cells were detected in thelungs of B7-H1-deficient mice as compared with WT mice (p<0.01; FIG.3A). On day 21-post tumor injection, the number of tumor metastases inthe lungs of naïve B7-H1-deficient mice was comparable to that of naïveWT mice (p=0.43; FIG. 3B). Fewer tumor metastases formed in the lungs ofimmunized WT mice as compared with naïve WT mice (p=0.001).Significantly, tumor metastases were completely rejected in the lungs ofimmunized B7-H1-deficient mice (FIG. 3B), suggesting that a moreefficient CD8+ T-cell memory population is established in the absence ofB7-H1.

Example 3—Bim Expression is Reduced in Antigen-Primed CD8+ T Cells inthe Absence of B7-H1

Studies were conducted to determine which mechanisms could beresponsible for the increased population of memory CD8+ T cells inB7-H1-deficient mice by examining the proliferation and apoptosis ofantigen-primed CD8+ T cells following immunization. CD11a was used as asurrogate activation marker. An advantage of this method is thatCD11a^(high) CD8+ T cells represent antigen-primed CD8+ T cells that areresponsive to undefined antigen epitopes not recognized by tetramers.CD11a^(high) CD8+ T cells were detected at low levels in the spleens ofnaïve WT and B7-H1-deficient mice (FIG. 4A). On day 7 afterimmunization, the percentage of CD11a^(high) CD8+ T cells increased morethan 2-fold in the spleens of B7-H1-deficient mice (41.5%) as comparedwith immunized WT mice (17.2%; p<0.01; FIGS. 4A and 4B), consistent withthe results obtained by tetramer staining (FIG. 1). Seven to 15% ofCD11a^(high) CD8+ T cells from WT and B7-H1-deficient mice were specificfor the known H-2K^(b)-restricted OVA₂₅₇₋₂₆₄ epitope based on tetramerstaining, and CD11a^(low) CD8+ T cells did not contain tetramer+ cells(FIG. 4C), suggesting that all antigen-specific CD8+ T cells are foundin the CD11a^(high) CD8+ T-cell population. In addition, theCD11a^(high) CD8+ population from both WT and B7-H1-deficient mice, butnot the CD11a^(low) CD8+ T-cell population, produced IFNγ and underwentdegranulation (indicated by CD107a surface expression) following ex vivore-stimulation (FIG. 4D). As T-cell responses against diverse epitopesare coordinately regulated, these data further support the concept thatthe CD11a^(high) CD8+ T-cell population represents true OVA-specificCD8+ T cells. Nearly 80-90% of OVA-induced CD11a^(high) CD8+ T cellswere reactive against undefined antigen epitopes of the OVA protein(FIG. 4C). Therefore, the CD11a^(high) CD8+ T-cell population could beused to represent a majority of the antigen-primed CD8+ T cells duringprimary T-cell responses. In the following studies, CD11a^(high) wasused as a marker to track antigen-specific CD8+ T cells.

The proliferation of effector CD8+ T cells following immunization wasexamined by staining cells for Ki67, a nuclear protein associated withcell proliferation (Gerdes et al. (1984) J. Immunol. 133:1710-1715). Thepercent of Ki67+ cells increased in CD11a^(high) CD8+ T cells fromB7-H1-deficient mice (9.32%) as compared with WT mice (7.5%), but thisincrease was not statistically significant (FIG. 5A). Proliferation alsowas monitored by performing a BrdU incorporation assay to measure theongoing proliferation of CD8+ T cells following immunization. In thisassay, the percentage of BrdU+ CD11a^(high) CD8+ T cells also wassimilar between WT (6.05%) and B7-H1-deficient mice (5.59%; FIG. 5B).Ki67+ or BrdU+ cells were mainly detected in the CD11a^(high) CD8+ Tcells but not in CD11a^(low) CD8+ T cells, suggesting that CD11a^(high)CD8+ T cells are proliferating following antigen-stimulation (FIGS. 5Aand 5B). These results suggested that the observed increased populationof antigen-primed CD8+ T cells in B7-H1-deficient mice is not due to anincreased proliferation of this cell compartment, as compared with WTmice.

Studies were then conducted to evaluate whether decreased apoptosis ofantigen-primed CD8+ T cells could contribute to the observed increasedpopulation of antigen-primed CD8+ T cells in immunized B7-H1-deficientmice. As discussed above, the Fas/Fas ligand death receptor pathway isimplicated in regulation of T-cell contraction, so the surfaceexpression levels of Fas and Fas ligand on effector CD8+ T cells weremeasured on day 7 after immunization. Expression of Fas and Fas ligandwas detected at similar levels in WT and B7-H1-deficient mice. Theseresults suggest that the observed increased population of effector CD8+T cells is not due to a change in Fas-induced apoptosis inB7-H1-deficient mice. The mitochondrial pathway for apoptosis wasinvestigated by analyzing levels of Annexin V and tetramethylrhodamineethyl ester (TMRE) staining. TMRE is a fluorescent marker that isincorporated into intact mitochondria, and cells undergoing apoptosisshow reduced TMRE staining as compared with live cells (Jayaraman, J.Immunol. Methods 306:68-79, 2005). These studies revealed that fewerantigen-primed CD11a^(high) CD8+ T cells were undergoing apoptosis(TMRE^(low) Annexin V+) in B7-H1-deficient mice (3.4%) as compared withWT mice (6.7%, p<0.05; FIGS. 5C and 5D). These results suggested thatdecreased levels of mitochondrial apoptosis may contribute to theobserved increased population of antigen-primed CD8+ T cells inB7-H1-deficient mice.

Experiments were conducted to look for alterations in the expression ofapoptosis-regulating molecules in effector CD8+ T cells. Intracellularlevels of the pro-apoptotic molecule Bim and the anti-apoptoticmolecules Bcl-2 and Bcl-x_(L) were measured in CD11a^(high) CD8+ T cellsfreshly isolated from the spleen on day 7 after immunization of naïvemice. Lower intracellular expression levels of Bim were observed inCD11a^(high) CD8+ T cells from B7-H1-deficient mice than in the samecells obtained from WT mice (p<0.001; FIGS. 6A and 6B), while theexpression levels of Bcl-2 and Bcl-x_(L) were comparable in WT andB7-H1-deficient mice (FIG. 6A). The expression of Bim, Bcl-2 andBcl-x_(L) were comparable in CD11a^(low) CD8+ T cells fromB7-H1-deficient and WT mice (FIG. 6A). Intracellular expression levelsof Bim also were analyzed in CD11a^(high) CD8+ T cells isolated from theliver on day 7 after immunization of naïve mice. Again, lowerintracellular expression levels of Bim were observed in CD11a^(high)CD8+ T cells from B7-H1-deficient mice as compared with WT mice (FIG.6C). Finally, intracellular expression levels of these proteins wereexamined in CD11a^(high) CD8+ T cells isolated from the spleen of naïvemice, and no significant differences were observed in B7-H1-deficientvs. WT mice in the expression levels of Bim (FIG. 6D), Bcl-2 orBcl-x_(L). These data suggested that the downregulation of thepro-apoptotic molecule Bim may contribute to the observed increasedpopulation of antigen-primed effector CD8+ T cells in B7-H1-deficientmice.

To exclude the possibility that the downregulation of Bim inB7-H1-deficient mice would be due to an intrinsic change inB7-H1-deficient T cells, transfer experiments were performed in whichnaïve OT-1 CD8+ T cells (Thy1.1+) were injected into WT orB7-H1-deficient mice (Thy1.2+). Following transfer of the OT-1 CD8+ Tcells, host mice were immunized with OVA plus poly I:C. On day 7 afterimmunization, the intracellular levels of Bim, Bcl-2, and Bcl-x_(L) weremeasured in transferred OT-1 CD8+ T cells freshly isolated from spleenand liver. OT-1 CD8+ T cells transferred into B7-H1-deficient hostsexpressed lower levels of Bim in both the spleen and liver as comparedwith the OT-1 CD8+ T cells transferred into WT hosts (FIG. 7). Theexpression of Bcl-2 and Bcl-x_(L) in OT-1 CD8+ T cells transferred intoWT or B7-H1-deficient mice was comparable (FIG. 7). These data suggestedthat the downregulation of Bim in B7-H1-deficient mice is not due to anintrinsic change in B7-H1-deficient T cells, but rather to host B7-H1interacting with one of its binding partners on CD8+ T cells.

Next, antibodies that block the interaction between B7-H1 and PD-1 orbetween B7-H1 and CD80 were used to examine if blocking either of thesepathways would impact Bim expression levels. On days 1 and 3 afterimmunization of WT mice with OVA plus poly I:C, an anti-PD-1 antibody(G4) that only blocks PD-1 binding to B7-H1 (Hirano et al. (2005) CancerRes. 65:1089-1096) or an anti-B7-H1 antibody (43H12) that only blocksB7-H1 binding to CD80 (Park et al. (2010) Blood 116:1291-1298) wasinjected. On day 7 after immunization, Bim expression levels inCD11a^(high) CD8+ T cells were compared between groups with or withoutantibody blockade. Antibodies blocking the interaction between B7-H1 andPD-1 or between B7-H1 and CD80 both reduced the expression of Bim inprimed CD8+ T cells as compared with control antibodies, whereas theexpression of Bcl-2 and Bcl-x_(L) remained unaffected. These resultssuggested that the downregulation of Bim in B7-H1-deficient mice mightbe due to a lack of interaction between B7-H1 and its binding partners,PD-1 and CD80.

After an acute viral infection, more central memory T cells accumulatein the lymphoid organs of PD-1-deficient mice as compared with WT mice,indicating that PD-1 signaling negatively regulates memory T-cellgeneration (Allie et al. (2011) J. Immunol. 186:6280-6286). Therelevance of CD80 signaling in the regulation of memory generation wasaddressed by transferring equal numbers of CD80-deficient OT-1 and WTOT-1 naïve CD8+ T cells into CD45.1+ mice. One day after T-celltransfer, host mice were immunized with OVA plus poly I:C. On day 21after immunization, the frequency and phenotype of the transferredCD80-deficient and WT OT-1 CD8+ T cells was analyzed. On day 21 afterimmunization, a 2-fold increased percentage of CD80-deficient OT-1 CD8+T cells as compared with WT OT-1 CD8+ T cells was detected in thespleen, indicating that the transferred CD80-deficient OT-1 CD8+ T cellsgenerated more memory T cells as compared with WT OT-1 CD8+ T cells.Surface staining confirmed that these cells had a central memoryphenotype (CD44^(hi) CD62L^(hi)). The recall response of the memorypopulation generated from transferred cells was investigated byinjecting the hosts with OVA plus poly I:C on day 30 after the initialimmunization, and 3 days later the frequency and phenotype of thetransferred cells was analyzed. An increased percentage ofCD80-deficient OT-1 CD8+ T cells as compared with WT OT-1 CD8+ T cellswas detected in the spleen (p=0.013). Surface staining confirmed thatthese cells had an effector memory phenotype (CD44^(hi)CD62L^(lo)).Taken together, these data demonstrated that Cd80−/− OT-1 CD8+ T cellsgenerated more memory T cells as compared their WT counterparts,indicating that CD80 expressed by CD8+ T cells may negatively regulatememory T-cell generation.

Example 4—B7-H1 Enhances Bim Expression in Activated CD8+ T Cells

Studies were conducted to investigate how B7-H1 might regulate Bimlevels in activated CD8+ T cells. Pre-activated WT CD8+ T cells wereincubated with platebound B7-H1 fusion protein for 48 hours in thepresence of TCR stimulation (anti-CD3 antibody). Bim expression wasanalyzed by western blotting, and increased expression levels wereobserved in CD8+ T cells cultured in the presence of B7-H1 fusionprotein, as compared with a control fusion protein (FIG. 8A). Bimexpression also was analyzed by intracellular flow cytometry, revealingthat the B7-H1 fusion protein dramatically increased the levels of Bimprotein in CD8+ T cells compared with a control fusion protein (p<0.02;FIGS. 8B and 8C). In the absence of anti-CD3 antibodies, Bim levels didnot increase upon incubation with B7-H1 fusion protein, suggesting thatB7-H1 provides a co-stimulatory signal for Bim upregulation.Accordingly, the absolute number of live cells was also reduced in CD8+T cells cultured in the presence of B7-H1 fusion protein compared with acontrol protein (p<0.01; FIG. 8D). Increased levels of cells undergoingapoptosis (TMRE^(low) Annexin V+) were observed in cultures of activatedCD8+ T cells exposed to the B7-H1 fusion protein and anti-CD3 (12.4%) ascompared with cells cultured with control fusion protein and anti-CD3(4.1%, FIG. 8E). The induction of apoptosis by B7-H1 fusion protein waslost in CD8+ T cells isolated from Bim-deficient and Bcl-2 transgenicmice (FIG. 8E), suggesting that B7-H1-induced T-cell apoptosis may bedependent on the Bim-mediated mitochondrial pathway of apoptosis.

To examine which receptor of B7-H1 is involved in mediating Bimupregulation, pre-activated WT CD8+ T cells were incubated withplate-bound B7-H1 fusion protein pre-blocked with anti-B7-H1 (10B5 or43H12) or anti-PD1 (G4) antibodies. The 10B5 antibody blocks theinteraction of B7-H1 with both PD-1 and CD80. Both 10B5 and G4antibodies completely blocked Bim upregulation induced by B7-H1 fusionprotein, while 43H12 only partially, but significantly, did so (FIG.8F). None of the antibodies used in this experiment had effects on Bimexpression levels in cells cultured with control fusion protein,indicating that their effect on Bim expression levels is due to blockingthe interaction between B7-H1/PD-1 or B7-H1/CD80, and not due to anon-specific effect. These results suggest that B7-H1 may use PD-1 orCD80 on CD8+ T cells to deliver co-stimulatory signals for theupregulation of Bim.

The mechanism by which B7-H1 regulates Bim expression levels was thenexamined. mRNA levels of Bcl2l11, which encodes the Bim protein, wereexamined by quantitative real-time PCR analysis using mRNA isolated frompre-activated CD8+ T cells that were exposed to B7-H1 fusion protein orto a control fusion protein and anti-CD3 for 24 hours. Incubation ofpre-activated CD8+ T cells with B7-H1 fusion protein did not increasethe levels of Bcl2l11 (FIG. 9A), indicating that the B7-H1-mediatedupregulation of Bim does not result from transcriptional regulation. Thedegradation of Bim is tightly regulated, at least in part via theactivation of Akt followed by Akt-mediated Bim phosphorylation anddegradation (Qi et al. (2006) J. Biol. Chem. 281:813-823). The level ofAkt activation in CD8+ T cells after B7-H1 engagement was measured byintracellular flow cytometry for phosphorylated-Akt (Ser473). CD8+ Tcells cultured with B7-H1 fusion protein exhibited decreased levels ofphosphorylated Akt as compared with CD8+ T cells cultured with a controlfusion protein (p<0.01; FIGS. 9B and 9C). As phosphorylation of Akt atSer473 is regulated by activation of mTOR (Sarbassov et al. (2005)Science 307:1098-1101; and Jacinto et al. (2006) Cell 127:125-137),studies were conducted to examine whether B7-H1 regulatesphosphorylation of mTOR in vitro. Unexpectedly, there was no differencein levels of phospho-mTOR in CD8+ T cells cultured with B7-H1 fusionprotein and cells cultured with control fusion protein (FIGS. 9B and9C). These results suggested that CD8+ T-cell engagement with B7-H1inhibits the activation of Akt, resulting in decreased degradation ofBim.

Example 5—Bim is Increased in Tumor-Reactive CD8+ T Cells in PeripheralBlood of Melanoma and Prostate Cancer Patients

Peripheral blood lymphocytes were isolated from 26 patients with stageIV (advanced) melanoma, and from 11 healthy blood donors. Lymphocyteswere stained with CD8, CD11a and PD-1 followed with intracellularstaining for Bim. High expression of CD11a by CD8 T cells was used toidentify antigen-primed T cells. Tumor-reactive CD8+ T cells weredefined by their expression of CD11a^(high) and PD-1+ (FIG. 13, leftpanel). The histograms shown in the right panel of FIG. 13 indicateexpression of Bim by subsets of CD8+ T cells (Tn: T naïve cells; PD-1−,PD-1 negative primed cells; PD-1+, PD-1 positive primed cells). Of note,only PD-1+ primed cells (tumor-reactive) CD8+ T cells expressed highlevels of Bim. Bim expression was increased in tumor-reactive CD8+ Tcells in peripheral blood of melanoma patients as compared to thehealthy controls, and also was increased in tumor-reactive CD8+ T cellsin peripheral blood of prostate cancer patients as compared to healthycontrols (FIG. 14). The Bim upregulation in melanoma patients was PD-1dependent, as depicted in FIG. 15. When levels of Bim were comparedbetween PD-1 negative (PD-1−) and PD-1 positive (PD-1+) CD11a^(high)CD8+ T cells, Bim was found to be significantly increased in the PD-1+populations (p=0.0081) in melanoma patients. In contrast, Bim expressionwas not increased in PD-1+ T cells in healthy donors, suggesting thatBim upregulation is dependent on PD-1 expression and is cancer-related.

Further, when melanoma patients were broken into “Bim low” vs. “Bimhigh” categories based on the level of Bim expression in tumor-reactivePD-1+ CD11a^(high) CD8+ T cells in the peripheral blood (FIG. 16, leftpanel), the survival rate for patients with Bim^(high) PD-1+ CD8+ Tcells was reduced as compared to the survival rate for patients with.Bim^(low) PD-1+CD8+ T cells (FIG. 16, right panel).

Example 6—B7-H1 Protein Induces High Expression of Bim in HumanPre-Activated CD8+ T Cells

Since Bim up-regulation is a consequence of interaction between B7-H1and PD-1, experiments were conducted to test whether an anti-PD-1blocking antibody can reduce B7-H1-induced Bim up-regulation in T cells.An in vitro system was established in which pre-activated human primaryCD8+ T cells were incubated with a B7-H1/PD-L1 fusion protein to induceBim up-regulation. As shown in FIG. 17A (right panel), a significantup-regulation of Bim (presented as MFI) was induced the B7-H1/PD-L1fusion (P<0.05, n=6). The increased Bim expression is furtherdemonstrated by a flow cytometry histogram (FIG. 17A, left panel) and aWestern blotting assay (FIG. 17B).

Using this system, several commercially available anti-human PD-1antibodies were screened for their blocking effects, and one anti-PD-1antibody (clone MIH4) was identified that significantly blockedB7-H1-induced Bim up-regulation in a dose dependent fashion (FIG. 18,left panel). Since B7-H1 induced different degrees of Bim up-regulationin individual healthy donors, experiments were conducted to examinewhether different degrees of Bim up-regulation would affect the blockingeffects of the anti-PD-1 antibody. Interestingly, it was observed thathigher levels of Bim induced by B7-H1 had a negative correlation withBim reduction by anti-PD-1 blocking antibody (FIG. 18, right panel;Pearson R=−0.71, n=12, P<0.05). These results suggested thatpre-existing Bim levels in CD8+ T cells might affect the efficiency ofanti-PD-1 blockade. Thus, measuring Bim levels before treatment couldhelp to determine the degree to which an anti-PD-1 antibody might blockthe impact of PD-1 signals on antitumor T cell responses.

Example 7—Anti-PD-1 Treatment Reduced the Frequency of Bim+ PD-1+Tumor-Reactive CD8 T Cells

Next, studies were conducted to evaluate the impact of the anti-PD-1antibody on Bim expression by tumor-reactive CD8 T cells in cancerpatients. Peripheral blood lymphocytes were collected from patients withadvanced melanoma (Stage IV) before and 12 weeks post anti-PD-1treatment. Tumor-reactive CD8 T cells were identified by their highexpression of CD11a and expression of PD-1. Bim expression was analyzedby intracellular staining. The percentage of Bim+PD-1+ in CD11a^(high)CD8+ T cells was compared between healthy people and melanoma patients,and between melanoma patients before and after treatment with anti-PD-1antibody. As shown in FIG. 19, the frequency of Bim+PD-1+ CD8 T cells inthe peripheral blood of melanoma patients (before treatment, n=29) wassignificantly higher than in the healthy control group (p=0.0012, n=14),suggesting that more PD-1+ CD8 T cells are under the influence of thePD-1/B7-H1 interaction that leads to up-regulation of Bim. Importantly,twelve weeks after anti-PD-1 antibody therapy (2 mg/kg, one cycle),about 67% (6/9) of the melanoma patients demonstrated a significantreduction in the frequency of Bim+PD-1+ CD8 T cells (p=0.023, n=6).These results indicated that measurement of Bim expressed by PD-1+ CD8 Tcells could be used to monitor the responses of cancer patients toanti-PD-1 therapy, which may block B7-H1-induced Bim up-regulation. Areduced frequency or level of Bim expressed by PD-1+ CD8 T cells incancer patients after anti-PD-1 therapy may be used to assess whichpatients are responsive to the therapy.

Example 8—B7-H1 Expressed by Tumor Cells Induces Bim Up-Regulation inHuman Pre-Activated CD8 T Cells

Since most human solid tumor cells express elevated levels of B7-H1, thefunction of tumor cell-expressed B7-H1 in T cell Bim expression wasexamined. Pre-activated human primary CD8 T cells were incubated withcells from a human melanoma line (624mel) that were transfected withB7-H1 cDNA or with control mock cDNA, for 24 hours. As shown in FIG. 20,intracellular expression of Bim was dramatically increased in CD8 Tcells cultured with B7-H1/624mel cells, as compared to mock/624mel cells(p<0.01). This result suggested that B7-H1 expressed by human tumorcells has the potential to up-regulate Bim in pre-activated CD8 T cells.

Example 9—Bim Expression is Associated with B7-H1 Expression in HumanRCC

The ability of B7-H1 to up-regulate Bim in pre-activated, but not newlyactivated, CD8+ T cells, implied that reactivation of tumor-reactiveCD8+ T cells at tumor sites could be dampened through this mechanism byB7-H1 positive tumor cells. To test this possibility, human cancertissues stained for B7-H1 and Bim were evaluated. The hypothesis wasthat B7-H1 positive human cancer tissues would be associated with moreBim positive tumor-infiltrating lymphocytes (TILs). As shown in FIG. 21(left panel), human renal cell carcinoma tissues were stained withanti-B7-H1 and anti-Bim antibodies in immunohistochemistry assays. B7-H1reactivity was identified on the surface of cancer cells, while Bimpositive staining was identified on cancer cells and also on TILs (FIG.21, left panel). Bim reactivity was determined by an arbitrary scoringsystem: 0 (absence), 1 (focal), 2 (moderate), and 3 (marked). Theassociation between B7-H1 positive or negative tumors and the frequencyof Bim reactivity at different levels is demonstrated in the right panelof FIG. 21, and was analyzed using Fisher's exact test. B7-H1 positivetumors were found in general to have a higher degree (2-3 scores) of Bimpositive TILs than B7-H1 negative tumors (Fisher's exact test, p<0.01).These results suggest that B7-H1 positive tumors can induce more deathin tumor-reactive T cells at tumor sites via up-regulation of Bim whenthese T cells are re-activated with tumor antigen stimulation.

Example 10—Bim Expression is Correlated with Granzyme B and T-BetExpressed by Cancer-Related PD-1+ CD11a^(high) CD8+ T Cells

To examine whether up-regulation of Bim is associated with effector Tcells, the levels of Granzyme B (an executive molecule of cytotoxic Tlymphocytes, CTL) and T-bet (a transcription factor of CTL) weremeasured in PD-1+ CD11a^(high) CD8 T cells from the blood of melanomapatients, and their correlation to Bim levels was analyzed. As shown inFIG. 22, positive correlations between levels of Bim and Granzyme B(left panel; r=0.51, p<0.05) and between levels of Bim and T-bet (rightpanel; r=0.62, p<0.01) were observed. These results suggested thathigher levels of Bim expression are associated with effector T celldifferentiation or function. These data also imply that up-regulation ofBim may be used by B7-H1 positive tumor cells to induce apoptosis oftumor-reactive CD8 T cells, especially of CD8 T cells with effectorfunction.

Example 11—Bim Expression Declines in PD-1+ CD11a^(high) CD8 T CellsFollowing Radiotherapy in Some Cancer Patients

To observe how the levels of Bim in tumor-reactive CD8 T cells respondto therapy, Bim levels were measured in PD-1+ CD11a^(high) CD8 T cellsfrom the peripheral blood of patients with melanoma and prostate cancersbefore and post radiotherapy. As shown in FIG. 23 (left panel),decreased levels of Bim were observed in melanoma patients postradiotherapy. In contrast, increased levels of Bim were observed inprostate cancer patients post radiotherapy (right panel). Due to thelimited numbers of patients in this study, these changes did not reachstatistical significance. However, these changes in Bim levels aftertumor cytotoxic therapy suggested that destruction of tumor tissuescould alter the antigen stimulation and B7-H1 expression that wouldresult in alterations in Bim expression, which is dependent on bothantigen stimulation and B7-H1 engagement with PD-1 on CD8 T cells. Takentogether, these studies indicate that measurement of Bim levels intumor-reactive PD-1+CD8 T cells could be used as a biomarker to monitorT cell responses to antigens and PD-1 ligands (e.g., B7-H1) expressed byhuman tumor cells.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. A method for treating a mammal having cancer,wherein said method comprises administering an anti-PD-1 antibody to amammal identified as containing an elevated level of Bim, and thusidentified as being likely to benefit from checkpoint blockade therapy,wherein said administering is under conditions wherein interaction ofnaturally-occurring B7-H1 with PD-1 in said mammal is reduced.
 2. Themethod of claim 1, wherein said mammal is a human.
 3. The method ofclaim 1, wherein said elevated level of Bim is based on Bim proteinlevels.
 4. The method of claim 1, wherein said elevated level of Bim isbased on the level of mRNA encoding Bim.
 5. The method of claim 1,wherein said cancer is a melanoma cancer, a breast cancer, a lungcancer, a renal cell carcinoma cancer, a pancreas cancer, a prostatecancer, a colon cancer, a brain cancer, a liver cancer, or an ovariancancer.